Abstract: Cross-linking chemical modification of galactose oxidase (GAO)) in the presence of substrate was carried out, and its substrate specificity was analysed. The substrate specificity of GAO was altered when cross-linking of GAO was made in the presence of galactose. The relative activity of native GAO for raffinose and melibiose were about 125 % and 80 % of the activity toward galactose, respectively. Tn contrast, the relative activity for raffinose and melibiose of GAO which was cross-linked in the presence of galactose showed only 90 % and 65 % of the activity toward galactose, respectively. Such alteration was not observed if cross-linked GAO was prepared in the absence of galactose. Kinetic analyses revealed that the effect of the presence of galactose during modification did not cause the difference in enzyme-substrate binding status in the ground status but decreased the stability of transition state of GAO- raffinose active complexTemplate and target information:bioimprinting